Thermo-Controllable High-Density Chips for Multiplex Analyses

ABSTRACT

The present invention provides miniaturized instruments for conducting chemical reactions where control of the reaction temperature is desired or required. Specifically, this invention provides chips and optical systems for performing and monitoring temperature-dependent chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost amplification of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 60/630,789 filed Nov. 24, 2004, and U.S. patentapplication Ser. No. 10/857,552 filed May 28, 2004, all of which arehereby incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to miniaturized instruments for conductingchemical reactions where control of the reaction temperature is desiredor required. Specifically, the invention relates to chips and opticalsystems for performing temperature-dependent chemical reactions. Theapparatus and methods embodied in the present invention are particularlyuseful for high-throughput and low-cost amplification of nucleic acids.

BACKGROUND OF THE INVENTION

The advent of Polymerase Chain Reaction (PCR) since 1983 hasrevolutionized molecular biology through vastly extending the capabilityto identify, manipulate, and reproduce DNA. Nowadays PCR is routinelypracticed in the course of conducting scientific researches, clinicaldiagnostics, forensic identifications, and environmental studies. PCRhas been automated through the use of thermal stable DNA polymerases anda machine commonly known as “thermal cycler”.

Performing a large quantity of PCR with the conventional thermal cyclerhas been rather expensive. This is partly due to the intrinsiclimitations of the conventional instrument. The conventional thermalcycler employs metal heating blocks and cooling reservoirs to carry outthe thermal cycling of reaction samples in plastic microfuge tubes.Because the instrument has a large thermal mass and the plastic tubeshave low heat conductivity, high level of electrical power is requiredto operate the instrument.

In recent years, the advancement in microfabrication technology enabledthe production of miniaturized devices integrated with electrical,optical, chemical or mechanical elements. The technology embodies arange of fabrication techniques including low-pressure vapor deposition,photolithography, and etching. Based on these techniques, miniaturizeddevices containing silicon channels coupled to micro-heaters have beenproposed (see, e.g., U.S. Pat. Nos. 5,639,423, 5,589,136 and 5,587,128).While the channel- or chamber-based design in principle reduces thethermal mass and the reaction volume, it still suffers from otherpractical drawbacks. In particular, the channels or chambers by designare not amenable to automated sealing that prevents evaporation and/orcross contamination of the reaction samples.

There thus remains a considerable need for small, mass produced, anddisposable devices designed to perform high-throughput amplification ofnucleic acids. A desirable device would allow (a) multiplexing anenormous quantity of amplification reactions; (b) automated and targetedsealing of the reaction sites on the devices; and (c) monitoring theprogress of the amplification reaction in real time. The presentinvention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

A principal aspect of the present invention is the design ofminiaturized devices applicable for multiplex analyses of individualmolecules, and/or simultaneous performance of a vast number of chemicalreactions. The devices and methods of the present invention simplify thelaborious and expensive procedures of performing chemical reactionswhere a control of the reaction temperature is desired or required.

Accordingly, in one embodiment, the present invention provides a chipfor varying and/or maintaining temperature of a reaction sample. Thechip comprises an array of thermo-controllable units that is in thermalcontact with a heating element, wherein the varying and/or maintainingof temperature is achieved by controlling the heating element alone, andwherein individual unit within the array comprises a micro well forreceiving and confining the reaction sample.

In another embodiment, the present invention provides a chip for varyingand/or maintaining a reaction sample that comprises an array ofthermo-controllable units, wherein the chip has a surface density of atleast about one thermo-controllable unit per 1 mm², and wherein a unitwithin the array comprises a micro well for receiving and confining thereaction sample, and a heating element in thermal contact with the microwell.

In yet another embodiment, the present invention provides a chip thatcomprises two arrays of thermo-controllable units, wherein one array isarranged in one orientation along the upper surface, and wherein theother array is arranged in an opposite orientation along the bottomsurface.

In a further embodiment, the present invention provides a chipcomprising an indium tin oxide heater (ITO-heater) in a glass plate thatis coupled to an array of thermo-controllable units fabricated in thechip.

In one aspect of these embodiments, the micro well is sealed uponfilling the reaction sample. Preferably, the well is sealed by (a)applying a radiation-curable adhesive along peripheral dimensionsdefining an open surface of the micro well; (b) placing a cover toencompass the peripheral dimensions that define the open surface of themicro well; and (c) exposing the micro well to a radiation beam toeffect the sealing. A wide range of radiation curable adhesive isapplicable for the present invention. They include but are not limitedto a diversity of acrylics, acrylates, polyurethanes (PUR), polyesters,vinyl, vinyl esters, and epoxies that are curable by radiation beamssuch as UV radiation and other radiation beams of various frequencies.

In certain aspects, the micro well in the subject chips are about 10 mmto about 100 μm in length, about 10 mm to about 100 μm in width, andabout 10 mm to about 100 μm in depth. The volume of the micro well isgenerally small, ranging from about 0.001 μl to about 100 μl. Wheredesired, not all of the thermo-controllable units within an array arefilled with reaction sample. Preferably, any two thermo-controllableunits filled with a reaction sample are separated by at least oneunfilled thermo-controllable unit. In certain aspects, the subject chipshave a surface density of at least one thermo-controllable unit per cm²,preferably at least about 10 per cm², or preferably at least about 100per cm², or preferably in the range of about 5 thermo-controllable unitsto about 50 thermo-controllable units per mm². Where desired, the chipsare operatively linked to a dispensing system for placing a reactionsample into the thermo-controllable units.

In other aspects, the thermo-controllable units of the subject chips canbe arranged in different temperature zones, each of which can beseparately controlled. In general, the micro well within each unit is inthermal contact with a heating element that can be made of metal orsemi-conducting material. Preferred heating element is anindium-tin-oxide (ITO) heater. Heating element can be located within themicro well, or can be attached to the upper or bottom, or both surfacesof the micro well. To prevent evaporation and condensation of thereaction sample on the upper surface of the well, the upper surface canbe maintained at a higher temperature than the bottom surface. Inpreferred embodiments, a plurality of grooves is fabricated to thebottom surface of the chip. The presence of the grooves greatlyfacilitates passive heat dissipation during thermal cycling. In otherembodiments, the subject chips have a ramp temperature time about 10° C.or higher per second, preferably about 25° C. or higher per second. Inother preferred embodiments, the subject chips may comprise temperaturesensors in thermal contact with the micro wells.

In certain embodiments, the subject chips are operatively coupled to anoptical system that detects optical signals emitted from the reactionsample. In preferred embodiments, the subject chips are fabricated withphoton-sensing elements in optical communication with the micro wellswhere chemical reactions are taking place. Representative photon-sensingelements include photo multiplier tube, charge coupled device, avalanchephoto diode, gate sensitive FET's and nano-tube FET's, and P-I-N diode.

The present invention also provides an apparatus for conducting achemical reaction requiring cycling at least two temperature levels. Theapparatus comprises a chip of the present invention and an opticalsystem that is operatively coupled to the chip. In this apparatus, theoptical system detects an optical signal such as luminescent signalcoming from the micro well fabricated in the chip. In one aspect, theoptical system monitors the optical signal over a multiple-cycle period.In another aspect, the optical system detects an optical signal that isproportional to the amount of product of the chemical reaction takingplace in the micro well over a multiple-cycle period. The optical systemcan include a spectrum analyzer that is composed of an opticaltransmission element and a photon-sensing element. Preferred opticaltransmission element can be selected from the group consisting ofmulti-mode fibers (MMF), single-mode fibers (SMF) and a waveguide.Preferred photon-sensing element can be selected from the groupconsisting of photo multiplier tube, charge coupled device, avalanchephoto diode, gate sensitive FET's and nano-tube FET's, and P-I-N diode.

In a preferred embodiment, the present invention provides an apparatusfor multiplexed analysis. The apparatus comprises an array of microwells for containing and confining reaction samples, wherein the arrayis optically linked to (a) an optical multiplexer adapted for receivingand multiplexing a plurality of incoming beams of one or more differentwavelengths; (b) an optical switch adapted for redirecting themultiplexed wavelengths of (a) to individual output fibers, wherein eachof the individual output fibers optically linked to a micro well of thearray, said micro well being coupled to a spectrum analyzer thatspectrally analyzes optical signals coming from the micro well.

The apparatus of the present invention is capable of performing a vastdiversity of chemical reactions. The subject apparatus is particularlysuited for performing enzymatic reactions, including but not limited tonucleic acid amplification reaction that encompasses PCR, quantitativepolymerase chain reaction (qPCR), nucleic acid sequencing, ligase chainpolymerase chain reaction (LCR-PCR), reverse transcription PCR reaction(RT-PCR), reverse transcription, and nucleic acid ligation.

Also provided by the present invention is a method of varying and/ormaintaining a temperature of a reaction sample. The method involves (a)placing the reaction sample into a micro well fabricated in a chip, saidmicro well being in thermal contact with a heating element, wherein thevarying and/or maintaining of temperature is achieved by controlling theheating element alone, and wherein the micro well receives and confinesthe reaction sample, and is sealed when filled with the reaction sample;(b) applying a voltage to the heating element thereby varying and/ormaintaining the temperature of the reaction sample. The step of applyinga voltage can be effected by processing a predetermined algorithm storedon a computer readable media operatively linked to the heating element.The method may also involve cycling the applying step to raise and lowerthe temperature of the reaction sample.

Further provided is a method of using a chip of the present invention toconduct a chemical reaction that involves a plurality of reactionsamples and requires cycling at least two temperature levels. The methodcomprises: (a) providing a subject chip; (b) placing the plurality ofreaction samples into the thermo-controllable units; and (c) controllingthe heating element to effect cycling at least two temperature levels.In one aspect of this embodiment, the controlling step comprisesprocessing sensor signals retrieved from a temperature sensoroperatively linked to a thermo-controllable unit based on protocolstored on a computer readable medium. In another aspect, the methodemploys a subject chip operatively coupled to an optical system thatdetects optical signals emitted from the reaction sample. In a preferredaspect, the optical system detects the optical signal without the needfor opening the micro well once the chemical reaction is initiated. Inyet another preferred aspect, the optical signals detected areproportional to the amount of product of the chemical reaction. Avariety of chemical reactions including a specific protein-proteininteraction and enzymatic reactions can be performed using this method.

The present invention also provides a method of using the subjectapparatus comprising the chip and optical system described herein todetect the presence or absence of a target nucleic acid in a pluralityof reaction samples. In this method, the formation of amplified targetnucleic acids indicates the presence of the target nucleic acid in thereaction sample. In certain aspects, the amplified target nucleic acidsare observed by transmitting excitation beams into the wells containingthe reaction samples, and detecting the optical signals coming from themicro well. In other aspects, formation of amplified target nucleicacids is observed by transmitting excitation beams into the wellscontaining the reaction samples at a plurality of times during theamplification, and monitoring the optical signals coming from the microwell at each of the plurality of times.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one illustrative chip design 101 having an integratedheating element and photon-sensing element.

FIG. 2 is a top view of one exemplary chip layout 101 on a circularsubstrate 201.

FIG. 3 is a schematic representation of one micro well on the chip.

FIG. 4 is a graph plotting the amount of time required to lose 10%reaction sample volume (y-axis) at various temperatures (x-axis). Threedifferent initial droplet volumes, namely 20 nl, 50 nl, and 100 nl areshown. The dew point under the test condition is about 14° C.

FIG. 5 depicts one illustrative chip design 501 having a sealed microwell. The micro well is sealed by first applying a UV-curable epoxyalong the peripheral dimensions that defines the open surface of themicro well, followed by laying a photo mask that allows penetration ofUV light along the peripheral dimensions, and then curing the epoxyusing the UV light.

FIG. 6A is a schematic longitudinal cross sectional view of an exemplarychip design according to the present invention. The chip comprisesseveral layers of materials, including a substrate layer fabricatedtherein a temperature sensor 604, a first heater 606, a photon-sensingdevice P-I-N diode 608. A waveguide 610, and a second heater arefabricated in the upper layers. The top layer comprises etched-ingrooves for placement of epoxy for purpose of sealing the micro well.FIG. 6B depicts an exemplary cover placed on top of the exemplary chipshown in 6A.

FIG. 7 is a schematic diagram showing different components of theoverall analysis system.

FIG. 8 is a top view of one exemplary chip design 1001. Shown in thefigure are 4 thermo-controllable units, each being surrounded at thebase by four L-shaped grooves (dashed lines). Each unit contains anintegrated heating element 1006.

FIG. 9 depicts a side view of the chip design of FIG. 8.

FIG. 10 is a bottom view of the chip design shown in FIG. 8.

FIG. 11 is a schematic longitudinal cross sectional view along thecutting line IV shown in FIG. 8.

FIG. 12 depicts a typical thermal cycling profile using an exemplarychip described herein.

FIG. 13 depicts another illustrative chip design of the presentinvention. The chip comprises two opposing arrays of thermo-controllableunits. Both arrays can be in thermal contact with a heater, one beingplaced on the upper surface, and another being placed at the bottomsurface of the chip.

FIG. 14 is a top view of an exemplary chip design adopting a 96-wellformat. Each well is optically linked to an optical transmissionelement, multi-mode fibers (MMF).

FIG. 15 depicts a side view of the chip shown in FIG. 14. The side viewshows a micro well optically linked to an optical system, having anoptical transmission element (e.g., multi-mode fibers (MMF)) andphoton-sensing element (e.g., CCD) on the top.

FIG. 16 depicts an apparatus having an array of micro wells opticallylinked to an optical system.

FIG. 17A depicts another apparatus of the present invention comprisingan array of thermo-controllable units optically linked to an opticalsystem. FIG. 17B depicts a top view of fiber bundle with localized 8-λexcitation light MMF and single 1 mm MMF for 8-λ different emissionlights in mixed format. This is an MMF bundled fiber sub-assembly withall central emission MMF fiber goes through AWG and rest of theexcitation to the sides.

FIG. 18 depicts a 12×8 fiber array each having 1λ8 channel and 18 mmlong DE-MUX AWG for passive low loss emission light separation with realtime analysis capability of all emissions simultaneously from a 16×96well chip. The spectrum can be resolved and analyzed by EMCCD.

FIG. 19 depicts another apparatus of the present invention comprising anarray of thermo-controllable units optically linked to an opticalsystem.

FIG. 20 depicts the SYBR Green-stained G6PDH gene products amplifiedusing a chip of the present invention.

FIG. 21 depicts the SYBR Green staining of G6PDH gene products appearedat the three different thermal stages of one PCR cycle. The three stagesare primer annealing at 45° C., denaturation of DNA at 95° C., andprimer-dependent extension at 72° C.

FIG. 22 depicts the amount of SYBR stain quantified throughout onecomplete thermal cycle.

FIG. 23 depicts the thermal cycle profiles of a chip of the presentinvention. The mean ramp rate is about 28° C. per second.

FIG. 24 depicts a chip with more than one temperature tone.

FIG. 25 depicts an exemplary method for sealing the subject micro well.

DETAILED DESCRIPTION OF THE INVENTION General Techniques:

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of Integrated Circuit (IC) processingbiochemistry, chemistry, molecular biology, genomics and recombinantDNA, which are within the skill of the art. See, e.g., Stanley Wolf etal., SILICON PROCESSING FOR THE VLSI ERA, Vols 1-4 (Lattice Press);Michael Quirk et al., SEMICONDUCTOR MANUFACTURING TECHNOLOGY; Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd)edition (1989); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R.Taylor eds. (1995).

Definitions

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

“Luminescence” is the term commonly used to refer to the emission oflight from a substance for any reason other than a rise in itstemperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when then move from an “excitedstate” to a lower energy state (usually the ground state); this processis often referred to as “radioactive decay”. There are many causes ofexcitation. If exciting cause is a photon, the luminescence process isreferred to as “photoluminescence”. If the exciting cause is anelectron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin-allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin-allowed transitions. Ifphotoluminescence is the result of a spin-forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” or “luminescentsignal” may have any one of the above-described properties.

A “primer” is a short polynucleotide, generally with a free 3′ —OHgroup, that binds to a target nucleic acid (or template) potentiallypresent in a sample of interest by hybridizing with the target nucleicacid, and thereafter promoting polymerization of a polynucleotidecomplementary to the target.

The terms “operatively linked to” or “operatively coupled to” are usedinterchangeably herein. They refer to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner.

Structure of the Chips of the Present Invention

A central aspect of the present invention is the design of miniaturizeddevices applicable for multiplexed analyses of individual molecules,and/or simultaneous performance of a vast number of chemical reactions.Distinguished from the previously reported micro-capillary devices orother channel-based instruments, the present invention provides a highlyautomated, miniaturized, analytical instrument that allows manipulationswith precise control of temperature, evaporation, small-volume reagentdelivery, product detection in a multiplexed fashion.

In one embodiment, the present invention provides chips for varyingand/or maintaining temperature of a reaction sample. The chips embodiedin the present invention generally comprise at least one array ofthermo-controllable units. The individual units within the array areseparated from each other by a physical barrier resistant to the passageof liquids. These units generally form indented areas referred to as“micro wells”. A micro well can be open at the top, but it must beenclosed otherwise and physically isolated from other wells to restrictpassage of liquids. Accordingly, the micro well has at least one cavitysuitable for receiving and confining reaction sample. By “confiningreaction sample” it is meant that the mobility of the reaction sample isrestricted by a physical barrier, and the sample is prevented fromflowing into another site once it is applied to the micro well of athermo-controllable unit.

The micro well may be fabricated in any convenient size, shape orvolume. The well may be about 10 mm to about 100 μm in length, about 10mm to 100 μm in width, and about 10 mm to 100 μm in depth. In general,the well has a transverse sectional area in the range of about 0.01 mm²to about 100 mm². The transverse sectional area may be circular,elliptical, oval, conical, rectangular, triangular, polyhedral, or inany other shape. The transverse area at any given depth of the well mayalso vary in size and shape.

The overall size of the chip ranges from a few microns to a fewcentimeters in thickness, and from a few millimeters to 50 centimetersin width or length. Preferred chips have an overall size of about 500micron in thickness and may have any width or length depending on thenumber of micro wells desired.

The cavity of each well may take a variety of configurations. Forinstance, the cavity within a micro well may be divided by linear orcurved walls to form separate but adjacent compartments, or by circularwalls to form inner and outer annular compartments.

The micro well typically has a volume of less than 500 ul, preferablyless than 50 ul. The volume may be less than 10 ul, or even less than 1ul. Where desired, the micro well can be fabricated in which the innersurface area to volume ratio is greater than about 1 mm²/1 ul, orgreater than 5 mm²/1 ul, or even greater than 10 mm²/l ul. Increasingthe surface area to volume ratio facilitates heat transfer through thethermal-controllable unit, thereby reducing the ramp time of a thermalcycle.

A micro well of high inner surface to volume ratio may be coated withmaterials to reduce the possibility that the reactants contained thereinmay interact with the inner surfaces of the well. Coating isparticularly useful if the reagents are prone to interact or adhere tothe inner surfaces undesirably. Depending on the properties of thereactants, hydrophobic or hydrophilic coatings may be selected. Avariety of appropriate coating materials are available in the art. Someof the materials may covalently adhere to the surface, others may attachto the surface via non-covalent interactions. Non-limiting examples ofcoating materials include silanization reagent such asdimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane ortrimethylchlorosilane, polymaleimide, and siliconizing reagents such assilicon oxide, Aquasil™, and Surfasil™. Additional suitable coatingmaterials are blocking agents such as amino acids, or polymers includingbut not limited to polyvinylpyrrolidone, polyadenylic acid andpolymaleimide.

The surface of the micro well can further be altered to createadsorption sites for reaction reagents. These sites may comprise linkermoieties for attachment of biological or chemical compound such as asimple or complex organic or inorganic molecule, a peptide, a protein(e.g. antibody) or a polynucleotide.

The total number of thermo-controllable units fabricated on the chipwill vary depending on the particular application in which the subjectchips are to be employed. To accommodate the need for simultaneousperformance of a vast number of reactions, the subject chips willgenerally comprise at least about 100 thermo-controllable units, usuallyat least about 500 thermo-controllable units, and more usually at leastabout 1000 units. The density of the thermo-controllable units on thechip surface may vary depending on the particular application.High-density chips usually contains on the surface at least about 10thermo-controllable units per cm², or preferably at least about 100thermo-controllable units per cm², or preferably in the range of about 5thermo-controllable units to about 50 thermo-controllable units per mm².

The thermo-controllable units of the subject chips can be arrayed in anyformat across or over the surface of the chip, such as in rows andcolumns so as to form a grid, in a circular pattern, and the like, see,e.g., FIG. 2. In a preferred embodiment, the thermo-controllable unitsare arrayed in a format compatible to instrumentation already exists fordispensing reagents and/or reading assays on microtiter plates. That wayextensive re-engineering of commercially available fluid handlingdevices is not required. Accordingly, the thermo-controllable units ofthe subject chips are preferably arranged in an 8×12 format. Using thisformat, each chip may have at least 96 thermo-controllable units,preferably at least 384 thermo-controllable units, more preferably atleast 1,536 units, and even more preferably 6,144 or 24,576 units. Whilethe number of thermo-controllable units of the 8×12 format chip may beas many as 24,576 or more, it usually does not exceed about 393,216units, and more usually does not exceed about 6,291,456 units.

The subject chips typically contain one or more grooves etched in at thebottom side of the chip. In general, the grooves are under-trenches,open channels or paths to allow air passage. The grooves reduce thethermal mass of the chip, increase the surface area, and thus enhancethe thermal performance of the chips. The grooves can be fabricated inany shapes, including but not limited to circular, elliptical, oval,conical, rectangular, triangular, and polyhedral. The grooves may befurther divided by linear or curved walls to form separate but adjacentchannels, or by circular walls to form inner and outer annular channels.The dimensions of the grooves will depend on the overall sizes anddepths of the chips. The depths of the grooves may range from about onetenth to about nine tenths of the chip depths. The other dimensions,namely widths and lengths, may be shorter, longer or comparable to thecorresponding dimensions of the chips. FIGS. 8-10 depict an exemplarygroove 1002 design. In particular, the L-shaped grooves surround thebase of a thermo-controllable unit. As the air flows through thepassageways formed by any of the grooves, it removes heat from thesurfaces of thermo-controllable unit by passive heat dissipation, thusincreasing the speed of thermal cycling.

The subject chips may contain more than one array of thermo-controllableunits. The arrays of thermo-controllable units may align horizontally,longitudinally, or diagonally long the x-axes or the y-axes of thechips. In addition, the arrays may align along curved walls within thechips that divide the arrays to form separate but adjacent compartments.

A preferred chip of the present invention comprises at least twoseparate arrays of thermo-controllable units. For instance, one arraymay be arranged in one orientation along the upper surface of the chip,and the another array may be arranged in an opposite orientation alongthe bottom surface of the chip. By “opposite orientation” it is meantthat one of the arrays is arranged in an inverted manner so that the topsurface of each thermo-controllable unit of the array (where thereagents can be added prior to sealing the surface) points away fromthat of the thermo-controllable units in the opposing array. The twoopposing arrays may be arranged such that the base of eachthermo-controllable unit is directly opposite to that of the opposingarray. Alternatively, the opposing arrays may be staggered such that thethermo-controllable units of the opposing arrays are located in betweenthose thermo-controllable units in the opposing array. FIG. 13 depictsan illustrative chip comprising at least two opposing arrays. In thisFigure, the chip 1301 has an upper 1302 and bottom surface 1304. One ofthe arrays is arranged along the upper surface 1306, and the other isarranged in an opposite orientation along the bottom surface 1308. Thethermo-controllable units of the bottom array are positioned in aninverted manner so that the open surface of each unit points away fromthat of the opposing unit in the chip. The two arrays are staggered sothat a cross-section line VII cuts through the thermo-controllable unitsfrom both the upper and the bottom arrays.

Though not specifically depicted in FIG. 13, any thermo-controllableunits in the upper and/or the bottom arrays may be sealed or unsealed.In addition, any thermo-controllable units within the upper and/or thebottom arrays may be filled or unfilled, with or without the reactionsample. Leaving one or more thermo-controllable units unfilled enhancesheat insulation because air is a poor conductor of heat. Accordingly, itis preferable to leave the adjacent thermo-controllable units empty soas to reduce heat transfer from one thermo-controllable unit to the nextunit. It is also preferable to leave the entire upper or the bottomarray of thermo-controllable units empty in order to minimize heattransfer from one layer to the next.

The subject array of thermo-controllable units is placed in thermalcontact with a heating element. This is achieved by integrating theheating element as part of the chip, or by placing the chip in contactwith an external heating element.

The heating element can be any heating means, including resistive heateror any other heat-generating device, which converts electrical orelectromagnetic energy into heat energy. Preferred heating elements aremicro-heaters compatible to the arrayed thermo-controllable units interms of size and configuration. The micro-heater can be placed as adetachable unit adjacent to, at the base and/or on top of the unit.Alternatively, the micro-heater can be affixed to the interior or theexterior surface of the thermo-controllable unit as an integral part ofthe unit. The integral heating element may surround the micro well wall,or located at the base of the thermo-controllable unit.

Micro-heaters are typically made of materials having high thermalconductivity and chemical stability. Such materials include but are notlimited to metals such as chromium, platinum and gold, andsemi-conductors such as ceramic, silicon, and geranium. A materialparticularly suitable for fabricating the micro-heaters is indium tinoxide (ITO). ITO is a transparent ceramic material with a very highelectrical conductivity. Because ITO can be prepared in bulk or in formof thin layer, it is particularly useful as either an integral or anexternal heating element.

The integral micro-heater generally is made of resistive heatingmaterial. Where the heating material is metal, it is generallypreferable to coat the surface with an insulating layer to preventcorrosion and/or electrophoresis of the sample components duringoperation with fluid samples. Coating is usually not necessary in caseof non-metal heating material. A variety of protective coatings areavailable, including those made of, e.g., SiO₂, Si₃N₄, and teflon. As isapparent to those skilled in the art, certain heating materials can bedeposited directly onto the substrate of the thermo-controllable units,and others may be deposited first onto an adhesion layer such astitanium or glass that in turn attaches to the substrate of the units.

The heating element is typically connected via electric leads to a powersource that provides voltage across the element and effects subsequentheating of the thermo-controllable units. The heating element may alsobe coupled to a temperature sensor that monitors and regulates thetemperature of the unit. The temperature sensor may control thetemperature and hence the thermal profile of an array ofthermo-controllable units. For instance, FIG. 24 depicts an exemplarychip with multiple temperature zones, in which each array of 96thermo-controllable units represents one temperature zone that iscontrolled by a temperature sensor and exhibiting a thermal profiledistinct from the rest of the temperature zones. Dividing the chip intovarious temperature zones provides additional flexibility for parallelperformance of chemical reactions that require different thermal cyclingprofiles. Alternatively, the temperature sensor can be coupled toindividual thermo-controllable unit so that the temperature of each unitcan be independently controlled. The temperature sensor may be includedas a detachable unit located adjacent to or at the base of thethermo-controllable unit. It can also be integrated into the interior orthe exterior surface of the unit. Furthermore, the temperature sensorcan be fabricated as an integral part of the micro-heater.

The subject chips can be provided with an optical system capable ofdetecting and/or monitoring the results or the progress of chemicalreactions taking place in the micro wells of the chips. Such opticalsystem achieves these functions by first optically exciting thereactants, followed by collecting and analyzing the optical signals fromthe reactants in the micro wells. The optical system applicable for thepresent invention comprises three elements, namely the opticalexcitation element, the optical transmission element, and thephoton-sensing element. The optical system may also comprise,optionally, an optical selection element.

The optical excitation element act as the source of excitation beamsused to optically excite the reactants contained in the micro wells.This element encompasses a wide range of optical sources that generatelight beams of different wavelengths, intensities and/or coherentproperties. Representative examples of such optical excitation sourcesinclude, but are not limited to, lasers, light-emitting diodes (LED),ultra-violet light bulbs, and/or white light sources.

The optical transmission element used in the present invention servestwo functions. First, it collects and/or directs the optical excitationsources to the reactants inside the micro wells of the chips. Second,it-transmits and/or directs the optical signals emitted from thereactants inside the micro wells of the chips to the photon-sensingelement. The optical transmission element suitable for use in thepresent invention encompasses a variety of optical devices that channellight from one location point to another. Non-limiting examples of suchoptical transmission devices include optical fibers, opticalmultiplexers (MUX) and de-multiplexers (DE-MUX), diffraction gratings,arrayed waveguide gratings (AWG), optical switches, mirrors, lenses,collimators, and any other devices that guide the transmission of lightthrough proper refractive indices and geometries.

The photon-sensing element analyzes the spectra of the optical signalscoming from the reactants inside the micro wells. Suitablephoton-sensing element can detect the intensity of an optical signal ata given wavelength, and preferably can simultaneously measure theintensities of optical signals across a range of wavelengths. Preferablythe element may also provide spectrum data analyses to show the spectrumpeak wavelength, spectrum peak width, and background spectrum noisemeasurements. Representative examples of suitable photon-sensing elementfor the present invention are avalanche photo diodes (APD),charge-coupled devices (CCD), electron-multiplying charge-coupled device(EMCCD), photo-multiplier tubes (PMT), photo-multiplier arrays, gatesensitive FET's, nano-tube FET's, and P-I-N diode. As used herein, CCDincludes conventional CCD, electron-multiplying charge-coupled device(EMCCD) and other forms of intensified CCD.

While the subject optical systems can be assembled using manycombinations of the various elements, a useful assembly for analyzingthe spectra of the excited reactants comprises an optical transmissionelement and a photon-sensing element. Such assembly is also referred toherein as “spectrum analyzer”.

Where desired, the optical system of the present invention can includean optical selection element. This element selects and/or refines theoptical properties of the excitation beams before they reaches thereactants contained in the micro wells. The optical selection elementcan also be employed to select and/or refine the optical signals comingfrom the reactants in the micro-wells before the signals reach thephoton-sensing element. Suitable optical selection element can selectand modify a wide range of optical properties, including but not limitedto, polarization, optical intensities, wavelengths, phase differencesamong multiple optical beams, time delay among multiple optical beams.Representative examples of such optical selection elements arepolarization filters, optical attenuators, wavelength filters (low-pass,band-pass or high-pass), wave-plates and delay lines.

The aforementioned optical elements can adopt a variety ofconfigurations. They can form integral parts of the subject chips orremain as separate units. All of these elements are commerciallyavailable. Accordingly, in one embodiment, the present inventionprovides a chip in which the optical transmission and photon-sensingelements are fabricated into the chip substrate. In one aspect, thephoton-sensing element is integrated into each micro well on the chipthat is to be monitored (see, e.g., FIGS. 1, 6A, and 11). In anotheraspect, more than one type of photon-sensing element is integrated intothe micro well to enhance the detection capability or efficiency. Inanother aspect, the photon-sensing element can be fabricated along theside or at the base of the micro well, or as part of the cover of themicro well. Photon-sensing elements suitable for such configurationinclude but are not limited to avalanche photo diode, charge coupleddevices (including conventional CCD, electron-multiplying charge-coupleddevice (EMCCD) and other forms of intensified CCD), gate sensitiveFET's, nano-tube FET's, P-I-N diode. Avalanche photo diode isparticularly preferred because it permits detections of a single photonby amplifying the signal through an avalanche process of electrontransfer. These elements together with the supporting circuitry can befabricated as part of the subject chips using standard IC processingtechniques described herein or known in the art.

In another embodiment, the present invention provides an apparatus inwhich the chip and the optical systems remain as separate units. Oneaspect of this embodiment encompasses an apparatus for conducting achemical or biological reaction requiring cycling at least twotemperature levels over a multiple-cycle period. The apparatus comprisesa chip of the present invention, and an optical system that isoperatively coupled to the chip and that detects an optical signalcoming from the micro well. Preferably, the optical signals detected arerelated to the amount of product of the chemical reaction taking placein the micro well.

FIG. 16 illustrates an exemplary optical system of this aspect. Thissystem includes an array of optical transmission element, namely the 1×L(where L is a positive integer) arrayed wavdguide 1606, that multiplexesup to L excitation beams 1610 into one optical beam 1612. The excitationbeams may have the same or different wavelengths ranging from, e.g., 200nm to 1000 nm. A plurality of M (where M is a positive integer) arrayedwaveguides, each channeling a multiplexed beam, are connected to an M×Noptical switch 1608 via the respective optical fiber 1616. The M×Noptical switch can direct M input excitation beams from the arrayedwaveguide 1606 to any one of its N output ports. Each of the N outputports is operatively coupled to a micro well through an optical fiber1604. Suitable optical fibers channeling the excitation beams to themicro well may include multi-mode fibers (MMF) and single-mode fibers(SMF). Upon excitation with the incident light beams, optical signalsare generated from the reactants inside the micro wells. These opticalsignals are then collected via an optical collimator 1614 to a 1×P(where P is a positive integer) arrayed waveguide 1616 whichde-multiplexes the optical signals. The de-multiplexed optical signalsare then transmitted to a spectrum analyzer, here a charge-coupleddevice (CCD) 1618 (which is part of the spectrum analyzer), for aspectrum analysis. CCDs having high number of pixels are preferred asthey provide a higher resolution of the optical signals being examined.

Another exemplary optical system of the present invention is depicted inFIG. 17. An array of fibers 1702 is employed to direct a plurality ofexcitation beams of the same or different wavelengths to a micro well1704 on a chip. The fibers within the array can be arranged in acircular configuration as shown in FIG. 17A or any other convenientconfigurations. The optical signals coming from the reactants in themicro well is then collected and transported by fibers 1706 to aspectrometer. The spectrometer periodically and sequentially samples andanalyzes the spectrum outputs of the fibers 1706. The optimal samplingfrequency can be empirically determined. It may range from once everymillisecond, to once every 150 milliseconds, and to once every 1500milliseconds. This configuration is particularly suited for a range ofspectroscopic applications because it permits the application of a widerange of excitation wavelengths to a reaction sample being examined. Assuch, the configuration supports analyses of fluorescence,chemiluminescence, scintillation, bioluminescence, and time-resolvedapplications without the need for frequent re-alignment of theexcitation sources that provide the appropriate excitation wavelengths.

FIG. 19 depicts another exemplary optical system of the presentinvention. In this system, optical fibers 1902 and beam collimators 1903connected to an excitation source 1901 are employed to illuminate allthe micro-wells on a chip 1904. The excitation source can be high powertunable lasers or Xenon lamps. The optical fibers 1902 are typicallymulti-mode fibers (MMF) of one millimeter diameter. The collimated beamsfrom the excitation source preferably provide uniform energydistribution across all the micro wells in the chip. The optical signalscoming from the micro wells on the silicon micro-plate are collimated bya lens 1905 and are passed through a tunable filter 1906 to anelectron-multiplying charge-coupled device (EMCCD) for spectrumanalysis. All of the elements including the optical fibers 1902,collimating lens 1903 and 1905, silicon micro-plate 1904, tunable filter1906 and EMCCD 1907, are enclosed in a highly protected dark housing1908. This particular embodiment offers a low cost solution formonitoring the progress and/or results of chemical reactions takingplace in micro wells fabricated on a chip.

Preparation of the Subject Chips

The chips of the present invention can be fabricated using techniqueswell established in the Integrated Circuit (IC) andMicro-Electro-Mechanical System (MEMS) industries. The fabricationprocess typically proceeds with selecting a chip substrate, followed byusing appropriate IC processing methods and/or MEMS micromachiningtechniques to construct and integrate various components.

Chip Substrate:

Several factors apply to the selection of a suitable chip substrate.First, the substrate must be a good thermal conductor. A good thermalconductor generally has a thermal conductivity value higher than 1 W/m⁻¹K⁻¹, preferably higher than 100 W/m⁻¹ K⁻¹, more preferably higher than140 W/m⁻¹ K⁻¹. Whereas the material's thermal conductivity may be 250W/m⁻¹ K⁻¹ or higher, it usually does not exceed 500 W/m⁻¹ K⁻¹. Second,the substrate must be relatively inert and chemically stable. Suchsubstrate generally exhibits a low level of propensity to react with thereaction samples employed in the intended application. Moreover, thematerials should also be selected based upon the ability or feasibilityto integrate the thermal control elements onto or adjacent to them. Avariety of materials meet these criteria. Exemplary materials includebut are not limited to metalloids or semiconductors, such as silicon,silicates, silicon nitride, silicon dioxide, gallium phosphide, galliumarsenide, or any combinations thereof. Other possible materials areglass, ceramics (including crystalline and non-crystalline silicate, andnon-silicate-based ceramics), metals or alloys, composite polymers thatcontain dopants (e.g., aluminum oxide to increase thermal conductivity),or any of a range of plastics and organic polymeric materials availablein the art.

Fabrication Process:

Fabrication of the subject chips can be performed according to standardtechniques of IC-processing and/or MEMS micromachining. Typically, thesubject chips are fabricated as multi-layer structures. The processgenerally proceeds with constructing the bottom layer. Then acombination of techniques including but not limited to photolithography,chemical vapor or physical vapor deposition, dry or wet etching areemployed to build structures located above or embedded therein. Vapordeposition, for example, enables fabrication of an extremely thin anduniform coating onto other materials, whereas etching allows for massproduction of larger chip structures. Other useful techniques such asion implantation, plasma ashing, bonding, and electroplating can also beemployed to improve the surface properties of the chips or to integratevarious components of the chips. The following details the fabricationprocess with reference to the exemplary chip designs depicted in thefigures. The same general process and the apparent variations thereofare applicable to fabricate any of the subject chips described herein.

FIG. 5 is a cross-section of an exemplary chip design 501. In thisembodiment, the micro well 502 is embedded within a body 504 which ismade up of first and second (or bottom and top) layers of substrates 506and 508, respectively. The process begins with providing a first layerof substrate which is generally a heat resistant material such as glass,Pyrex wafer, or any other suitable materials described herein or knownin the art. The next step is to create the micro well 502 that forms thebasis of the thermo-controllable unit. The micro well is generallydisposed within the second layer 508 that is typically a silicon wafer.The silicon wafer may go through several processing steps prior to beingattached to the first layer. For example, the silicon wafer may beattached to a layer of photoresist to render the surface moresusceptible to chemical etching after exposure to UV light during theprocess of photolithography. The layer of photoresist defines, byprecise alignment of the photo-mask, the size and location of the microwell that is to be formed by a subsequent etching step. The siliconwafer is then etched by a variety of means known in the art to form thewell cavity. A commonly practiced etching technique involves the use ofchemicals, e.g., potassium hydroxide (KOH), which removes the siliconwafer to form the desired shape.

The heating element described herein can be deposited onto the interiorsurface of the micro well (see, e.g., FIGS. 6A and 11). Themicro-heaters, for example, may be arranged to surround the micro wellwall, or form the base of the micro well. The micro-heater and the fluidcontained in the well can be isolated electrically and chemically fromeach other by an insulating or protective coating. Coating isparticularly preferable in case of metal heating element that may beprone to corrosion and/or electrophoresis of the sample componentsduring operation with fluid samples. A variety of protective coatingsare available in the art, including those made of, e.g., SiO₂, Si₃N₄,and Teflon. Where the heating element is indium tin oxide, it ispreferable to use glass (e.g. on borosilicate glass), quartz, or thelike material as the adhesion layer before depositing it into the microwell.

Integrated circuitry that supports the operations of the heating elementand/or the temperature sensor can also be implanted into the well oronto the exterior part of the silicon layer by a suitable IC-processingtechnique described herein or known in the art.

The second layer of silicon 508 in FIG. 5 or other suitable substratematerial can be attached to the first glass layer in one of severalways. Anodic bonding can be used when the materials employed arecompatible with the bonding requirements. Alternatively, a polymericbonding compound such as benzocyclobutene (BCB) (available from DowChemical) can be applied to adhere one layer onto the next. In addition,the two layers of substrates can be fused together by extensive heatingunder high temperatures.

FIG. 6 and FIGS. 8-11 depict other exemplary chip designs 601 in whichthe first layer is made of silicon or the like material. In FIGS. 6 and11, the temperature sensors 604 and 1111, heating elements 606 and 1006,and photo-sensing elements 608 and 1010 are fabricated in the firstlayer using methods described above or other methods illustrated in thepending application Ser. No. 10/691,036, the content of which isincorporated by reference in its entirety.

To enhance the detection and sensing capabilities of the chip,additional layers of sensing structures such as waveguides 610 arefabricated. The waveguides are constructed to channel light beamsemitted from one or multiple micro wells through a side wall of themicro well 610. While it may be preferable to couple one waveguide to asingle micro well to effect separate detection of light signals emittedfrom individual wells, channeling signals from multiple wells arepossible by adjusting the excitation light beam. For instance, theincoming light can be synchronized in or out of phase with light signalscollected from other waveguides such that multiple pulses of light beamsof known wavelengths and intensities arrive at different micro wellswithin predetermined time frames. The sensor reading associated withthat particular light pulse is then monitored with appropriate postprocessing. The materials with which the waveguides are fabricated aredetermined by the wavelength of the light being transmitted. Silicondioxide is suitable for transmitting light beams of a wide spectrum ofvisible wavelengths. Silicon and polysilicon are applicable for guidinginfra-red wavelengths. Those skilled in the art will know of othermaterials suitable for constructing waveguide. To achieve the desiredpolarization states, waveguides with appropriate integral gratings canbe constructed using standard MEMS micromachining techniques.

The chip depicted in FIG. 6A also contains a top layer micro-heater. Thetop layer heater provides an additional source of heat energy to effecta rapid thermal cycling. It may also serve as a physical barrier toprevent evaporation of the reaction reagents applied to the micro well.To further minimize evaporation, the top layer heater can be maintainedat a higher temperature, usually a few Celsius degrees higher relativeto the bottom heater. The type of heater to be placed on the uppersurface will depend on the intended use of the chip. Indium tin oxideheater is generally preferred because it is transparent to visiblelight. When deposited on glass and applied to the top of the chip, lightemitted from the micro well can still pass through and be detected by aphoton-sensing element.

Once the micro wells of the subject chips are fabricated, their surfaceproperties can be improved to suit the particular application. Wherelarge surface area is desired, the wall of the micro well may be furtheretched by, e.g., a plasma etcher to obtain very fine dendrites ofsilicon, commonly referred to as “black silicon”. The presence of blacksilicon can dramatically increase the effective heating surface area.The black silicon fabricated at the base of the micro well may alsoserve as an anchor for photon-sensing devices, temperature sensors andother control elements.

As discussed in the sections above, a micro well of high inner surfaceto volume ratio may be coated with materials to reduce the possibilitythat the reactants contained therein may interact with the innersurfaces of the well. The choice of methods for applying the coatingmaterials will depend on the type of coating materials that is used. Ingeneral, coating is carried out by directly applying the materials tothe micro well followed by washing the excessive unbound coatingmaterial. Certain coating materials can be cross-linked to the surfacevia extensive heating, radiation, and by chemical reactions. Thoseskilled in the art will know of other suitable means for coating a microwell fabricated on chip, or will be able to ascertain such, withoutundue experimentation.

The surface of the micro well can further be altered to createadsorption sites for reaction reagents. One skilled in the art willappreciate that there are many ways of creating adsorption sites toimmobilize chemical or biological reactants. For instance, a wealth oftechniques are available for directly immobilizing nucleic acids andamino acids on a chip, anchoring them to a linker moiety, or tetheringthem to an immobilized moiety, via either covalent or non-covalent bonds(see, e.g., Methods Mol. Biol. Vol. 20 (1993), Beier et al., NucleicAcids Res. 27:1970-1-977 (1999), Joos et al., Anal. Chem. 247:96-101(1997), Guschin et al., Anal. Biochem. 250:203-211 (1997)).

The subject chips can be further modified to contain one or more grooveson the top, or at the bottom side of the chip (see, e.g., FIGS. 6A and9). Grooves are generally fabricated by etching the bottom side siliconwafer. Back-side etching can be carried out before or after formation ofthe micro well.

Sealing Process

In most of the applications, sealing the micro wells is desirable toprevent evaporation of liquids and thus maintains the preferred reactionconcentrations throughout the thermal cycling. Accordingly, the presentinvention provides a technique for sealing an array of micro wells. Thedesign of the subject sealing technique takes several factors intoconsideration. First, the technique should be amenable to highthroughout processing of a large quantity of micro wells. Second, themethod should permit selective sealing of individual micro wells. Assuch, the method can yield chips comprising open micro wellsinterspersed among sealed micro wells in any desired pattern or format.As mentioned above, chips having both open and sealed micro wells areparticularly desirable. The open and/or unfilled wells not only allowpassive dissipation of heat, but also reduce heat transfer between theneighboring micro wells.

A preferred method of sealing an array of micro wells containing atleast one open well. The method comprises the steps of (a) applying aradiation-curable adhesive along peripheral dimensions defining the opensurface of the at least one open micro well; (b) placing a cover toencompass the peripheral dimensions that define the open surface of theat least one open micro well that is to be sealed; and (c) exposing thearray to a radiation beam to effect the sealing.

As used herein, “radiation-curable adhesive” refers to any compositionthat cures and bonds to the adhering surface upon exposure to aradiation beam without the need of extensive heating. “Radiation beam”refers to electromagnetic waves of energy including, in an ascendingorder of frequency, infrared radiation, visible light, ultraviolet (VU)light, X-rays, and gamma rays. A vast number of radiation-curableadhesive are commercially available (see, e.g., a list of companiesselling radiation-curable adhesive and radiation systems fromThomasNet®'s worldwide web site). They include a diversity of acrylics,acrylates, polyurethanes (PUR), polyesters, vinyl, vinyl esters, and avast number of epoxies that are curable by radiation beams at variousfrequencies. These and other radiation-curable materials are suppliedcommercially in form of liquid, or solid such as paste, powder, resin,and tape.

The choice of radiation-curable adhesive will dependent on the materialsmade up the surfaces to be adhered. The aforementioned classes ofadhesive are suited for adhering the chip substrate to the cover whichcan be made of a range of materials. For instance, acrylics and epoxiesare applicable for radiation-sealing any two surfaces, made of any oneof the materials selected from glass, ceramics, metalloids,semiconductors (e.g., silicon, silicates, silicon nitride, silicondioxide, quartz, and gallium arsenide), plastics, and other organicpolymeric materials. Radiation-curable materials exhibiting theproperties of low use temperature and rapid curing time are particularlydesirable for sealing the subject chips. These materials allow for arapid sealing to avoid radiation damages to the chemical or biologicalreagents contained in the chips.

The radiation-curable adhesive can be applied by any mechanical meansalong the peripheral dimensions that define the open surface of a microwell. The “peripheral dimensions” can be the boundaries on the chipsubstrate or on the cover. In either case, the peripheral dimensionsbecome bonded to the respective adhering surface, the substrate or thecover, upon curing the adhesive. The radiation-curable adhesive can besmeared, printed, dispensed, or sprayed onto the peripheral dimensionsusing any suitable tools. Preferred mechanical means yields a uniformlayer of adhesive on the peripheral dimensions. One way to provide auniform distribution is to apply the adhesive directly onto theperipheral dimensions of an open well using a squeegee over a meshedscreen mask (see, e.g., FIG. 25). Alternatively, the radiation-curableadhesive can be applied directly onto the cover that has been markedwith the peripheral dimensions using the meshed screen mask. A uniformlayer of adhesive is achieved upon removal of the mask.

Upon application of the radiation-curable adhesive, a cover is placed onthe micro well to encompass the peripheral dimensions that define theopen surface of the well. Suitable covers are generally made ofmaterials that permit passage of a radiation beam. Preferred covers arefabricated with transparent materials such as glass, quartz, plastic,any suitable organic polymeric materials known to those skilled in theart, or any combinations thereof.

Sealing a covered micro well is carried out by exposing the well to aradiation beam. Depending on the type of adhesive selected, theradiation beam may come from a conventional incandescent source, alaser, a laser diode, UV-bulb, an X-ray machine or gamma-ray machine, orthe like. Where desired, radiation beam from the radiation source ispermitted to reach only selected locations on the micro well array sothat only certain selected wells are to be sealed. A selective sealingis often achieved by using a photo-mask patterned with the locations ofthe micro wells. The photo-mask is provided with transparent locationsand opaque locations that correspond to the micro wells that are to besealed and those that are to remain open, respectively. The radiationbeam passes freely through the transparent regions but is reflected fromor absorbed by the opaque regions. Therefore, only selected micro wellsare exposed to light and hence sealed by curing the adhesive. In oneembodiment, the photo-mask is patterned such that no two adjoining openmicro wells are to be sealed. In another embodiment, the photo-mask ispatterned such that the resulting micro well array contains alternatingsealed and unsealed wells. One skilled in the art can fashion anunlimited number of photo-masks with any patterns to yield chipscontaining open and sealed micro wells in any format. Methods formanufacturing such photo-masks are well established in the art and henceare not detailed herein.

Uses of the Subject Chip and Other Devices of the Present Invention

The subject chips have a wide variety of uses in chemical and biologicalapplications where controllable temperatures are desired.

In one embodiment, the subject chips can be used to vary and/or maintaintemperature of a reaction sample. Varying and/or maintaining temperatureof a reaction sample are required in a wide range of circumstancesincluding but not limited to discerning protein-protein interaction,examining DNA or RNA hybridization, and performing enzymatic reaction.The method involves placing the reaction sample into a micro wellfabricated in a chip that is in thermal contact with a heating element,and applying a voltage to the heating element.

In another embodiment, the subject chips are used for conducting achemical reaction that involves a plurality of reaction samples andrequires cycling at least two temperature levels. The process involves(a) providing a chip comprising an array of thermo-controllable units asdescribed herein; (b) placing the plurality of reaction samples into thethermo-controllable units of the chip; and (c) controlling the heatingelement to effect cycling at least two temperature levels.

As used herein, the term “chemical reaction” refers to any processinvolving a change in chemical properties of a substance. Such processincludes a vast diversity of reactions involving biological moleculessuch as proteins, glycoproteins, nucleic acids, and lipids, or inorganicchemicals, or any combinations thereof. The chemical reaction may alsoinvolve interactions between nucleic acid molecules, between proteins,between nucleic acid and protein, between protein and small molecules.Where the process is catalyzed by an enzyme, it is also referred to as“enzymatic reaction.”

The subject chips and other apparatus are particularly useful inconducting enzymatic reactions because most enzymes function under onlycertain temperatures. Representative enzymatic reactions that areparticularly temperature dependent include but are not limited tonucleic acid amplification, quantitative polymerase chain reaction(qPCR), nucleic acid sequencing, reverse transcription, and nucleic acidligation.

Practicing the subject method generally proceeds with placing thereaction sample into a micro well of the subject chip that is in thermalcontact with a heating element. Where desired, the reaction sample canbe applied by a dispensing system operatively coupled to the subjectchip. A variety of dispensing instruments, ranging from manuallyoperated pipettes to automated robot systems are available in the art.Preferred dispensing instruments are compatible to the particular format(e.g. 96-well) of the subjects chip.

To prevent evaporation of aqueous reaction samples, the samples can beapplied to the micro well at or around dew point. As used herein, “dewpoint” refers to a temperature range where the droplet size does notchange significantly. At dew point, an equilibrium is reached betweenthe rate of evaporation of water from the sample droplet and the rate ofcondensation of water onto the droplet from the moist air overlying thechip. When this equilibrium is realized, the air is said to be saturatedwith respect to the planar surface of the chip. At one atmosphericpressure, the dew point is about 14° C. Accordingly, dispensing aqueousreaction samples is preferably carried out at a temperature no more thanabout 1° C. to about 5° C. degrees above dew point. As is apparent toone skilled in the art, dew point temperature increases as the externalpressure increases. Therefore, where desired, one may dispense thereaction samples in a pressured environment to prevent evaporation.

In practice, controlling the heating element and hence the temperatureof the reaction sample, is effected by processing a predeterminedalgorithm stored on a computer readable medium operatively linked to theheating element. In other aspects, the controlling step may involveprocessing sensor signals retrieved from a temperature sensor elementthat is operatively linked to a thermo-controllable unit based onprotocols stored on a computer readable medium. This can be achieved byemploying conventional electronics components for temperature controlthat may process either analog or digital signals. Preferably, theelectronics components are run on a feedback control circuitry. They cancontrol the temperature of one unit, but more often the temperature ofmultiple thermo-controllable units that collectively form onetemperature zone. Where desired, the chemical reactions can take placein different thermo-controllable units located in different temperaturezones. In certain embodiments, the temperatures of the different zonesare separately controlled. The thermal cycling profile and duration willdepend on the particular application in which the subject chip is to beemployed.

Nucleic Acid Amplification:

The chips of the present invention provide a cost-effective means foramplifying nucleic acids. Unlike the conventional thermal cyclers, thesubject chips are highly miniaturized, capable of performing rapidamplification of a vast number of target nucleic acids in small volume,and under independent thermal protocols.

As used herein, “nucleic acid amplification” refers to an enzymaticreaction in which the target nucleic acid is increased in copy number.Such increase may occur in a linear or in an exponential manner.Amplification may be carried out by natural or recombinant DNApolymerases such as Taq polymerase, Pfu polymerase, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, and/or RNA polymerases suchas reverse transcriptase.

A preferred amplification method is polymerase chain reaction (PCR).General procedures for PCR are taught in U.S. Pat. No. 4,683,195(Mullis) and U.S. Pat. No. 4,683,202 (Mullis et al.). Briefly,amplification of nucleic acids by PCR involves repeated cycles ofheat-denaturing the DNA, annealing two primers to sequences that flankthe target nucleic acid segment to be amplified, and extending theannealed primers with a polymerase. The primers hybridize to oppositestrands of the target nucleic acid and are oriented so that thesynthesis by the polymerase proceeds across the segment between theprimers, effectively doubling the amount of the target segment.Moreover, because the extension products are also complementary to andcapable of binding primers, each successive cycle essentially doublesthe amount of target nucleic acids synthesized in the previous cycle.This results in exponential accumulation of the specific target nucleicacids at approximately a rate of 2^(n), where n is the number of cycles.

A typical conventional PCR thermal cycling protocol comprises 30 cyclesof (a) denaturation at a range of 90° C. to 95° C. for 0.5 to 1 minute,(b) annealing at a temperature ranging from 55° C. to 65° C. for 1 to 2minutes, and (c) extension at 68° C. to 75° C. for at least 1 minutewith the final cycle extended to 10 minutes. With the subject chips, thethermal cycling time can be drastically reduced because of, partly, thesmall reaction volume, the small heating mass, and the design ofeffective heat dissipation features.

A variant of the conventional PCR is a reaction termed “Hot Start PCR”.Hot Start PCR techniques focus on the inhibition of polymerase activityduring reaction preparation. By limiting polymerase activity prior toPCR cycling, non-specific amplification is reduced and the target yieldis increased. Common methods for Hot Start PCR include chemicalmodifications to the polymerase (see, e.g., U.S. Pat. No. 5,773,258),inhibition of the polymerase by a polymerase-specific antibody (see,e.g., U.S. Pat. No. 5,338,671), and introduction of physical barriers inthe reaction site to sequester the polymerase before the thermal cyclingtakes place (e.g., wax-barrier methods). The reagents necessary forperforming Hot Start PCR are conveniently packaged in kits that arecommercially available (see, e.g., Sigma's JumpStart Kit).

Another variant of the conventional PCR that can be performed with thesubject chips is “nested PCR” using nested primers. The method ispreferred when the amount of target nucleic acid in a sample isextremely limited for example, where archival, forensic samples areused. In performing nested PCR, the nucleic acid is first amplified withan outer set of primers capable of hybridizing to the sequences flankinga larger segment of the target nucleic acid. This amplification reactionis followed by a second round of amplification cycles using an inner setof primers that hybridizes to target sequences within the large segment.

The subject chips can be employed in reverse transcription PCR reaction(RT-PCR). RT-PCR is another variation of the conventional PCR, in whicha reverse transcriptase first coverts RNA molecules to double strandedcDNA molecules, which are then employed as the template for subsequentamplification in the polymerase chain reaction. In carrying out RT-PCR,the reverse transcriptase is generally added to the reaction sampleafter the target nucleic acids are heat denatured. The reaction is thenmaintained at a suitable temperature (e.g., 30-45° C.) for a sufficientamount of time (e.g., 5-60 minutes) to generate the cDNA template beforethe scheduled cycles of amplification take place. Such reaction isparticularly useful for detecting the biological entity whose geneticinformation is stored in RNA molecules. Non-limiting examples of thiscategory of biological entities include RNA viruses such as HIV andhepatitis-causing viruses. Another important application of RT-PCRembodied by the present invention is the simultaneous quantification ofbiological entities based on the mRNA level detected in the test sample.One of skill in the art will appreciate that if a quantitative result isdesired, caution must be taken to use a method that maintains orcontrols for the relative copies of the amplified nucleic acids.

Methods of “quantitative” amplification of nucleic acids are well knownto those of skill in the art. For example, quantitative PCR (qPCR) caninvolve simultaneously co-amplifying a known quantity of a controlsequence using the same primers. This provides an internal standard thatmay be used to calibrate the PCR reaction. Other ways of performing qPCRare available in the art and are detailed in the section “Detection ofAmplified Target Nucleic Acids” below.

The subject chips can also be employed to form ligase chain polymerasechain reaction (LCR-PCR). The method involves ligating the targetnucleic acids to a set of primer pairs, each having a target-specificportion and a short anchor sequence unrelated to the target sequences. Asecond set of primers containing the anchor sequence is then used toamplify the target sequences linked with the first set of primers.Procedures for conducting LCR-PCR are well known to artisans in thefield, and hence are not detailed herein (see, e.g., U.S. Pat. No.5,494,810).

Nucleic acid amplification is generally performed with the use ofamplification reagents. Amplification reagents typically includeenzymes, aqueous buffers, salts, primers, target nucleic acid, andnucleoside triphosphates. Depending upon the context, amplificationreagents can be either a complete or incomplete amplification reactionmixture.

The choice of primers for use in nucleic acid amplification will dependon the target nucleic acid sequence. Primers used in the presentinvention are generally oligonucleotides, usually deoxyribonucleotidesseveral nucleotides in length, that can be extended in atemplate-specific manner by the polymerase chain reaction. The design ofsuitable primers for amplifying a target nucleic acid is within theskill of practitioners in the art. In general, the following factors areconsidered in primer design: a) each individual primer of a pairpreferably does not self-hybridize; b) the individual pairs preferablydo not cross-hybridize; and c) the selected pair must have theappropriate length and sequence homology in order to anneal to twodistinct regions flanking the nucleic acid segment to be amplified.However, not every nucleotide of the primer must anneal to the templatefor extension to occur. The primer sequence need not reflect the exactsequence of the target nucleic acid. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer with theremainder of the primer sequence being complementary to the target.Alternatively, non-complementary bases can be interspersed into theprimer, provided that the primer sequence has sufficient complementarilywith the target for annealing to occur and allow synthesis of acomplementary nucleic acid strand.

For a convenient detection of the amplified nucleotide acids resultingfrom PCR or any other nucleic acid amplification reactions describedabove or known in the art, primers may be conjugated to a detectablelabel. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means. Awide variety of appropriate detectable labels are known in the art,which include luminescent labels, enzymatic or other ligands. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as digoxigenin, ß-galactosidase, urease,alkaline phosphatase or peroxidase, avidin/biotin complex.

The labels may be incorporated by any of a number of means well known tothose of skill in the art. In one aspect, the label is simultaneouslyincorporated during the amplification step. Thus, for example, PCR withlabeled primers or labeled nucleotides can provide a labeledamplification product. In a separate aspect, transcription reaction inwhich RNA is converted into DNA, using a labeled nucleotide (e.g.fluorescein-labeled UTP and/or CTP) or a labeled primer, incorporates adetectable label into the transcribed nucleic acids.

The primer pairs used in this invention can be obtained by chemicalsynthesis, recombinant cloning, or a combination thereof. Methods ofchemical polynucleotide synthesis are well known in the art and need notbe described in detail herein. One of skill in the art can use thetarget sequence to obtain a desired primer pairs by employing a DNAsynthesizer or ordering from a commercial service.

Nucleic acid amplification requires a target nucleic acid in a buffercompatible with the enzymes used to amplify the target. The targetnucleic acid used for this invention encompasses any reaction samplessuspected to contain the target sequence. It is not intended to belimited as regards to the source of the reaction sample or the manner inwhich it is made. Generally, the test sample can be biological and/orenvironmental samples. Biological samples may be derived from human,other animals, or plants, body fluid, solid tissue samples, tissuecultures or cells derived therefrom and the progeny thereof, sections orsmears prepared from any of these sources, or any other samplessuspected to contain the target nucleic acids. Preferred biologicalsamples are body fluids including but not limited to blood, urine,spinal fluid, cerebrospinal fluid, sinovial fluid, ammoniac fluid,semen, and saliva. Other types of biological sample may include foodproducts and ingredients such as vegetables, dairy items, meat, meatby-products, and waste. Environmental samples are derived fromenvironmental material including but not limited to soil, water, sewage,cosmetic, agricultural and industrial samples.

Preparation of nucleic acids contained in the test sample can be carriedout according to standard methods in the art or procedures described.Briefly, DNA and RNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (“Molecular Cloning: A Laboratory Manual”), or extracted by nucleicacid binding resins following the accompanying instructions provided bymanufacturers' instructions.

The nucleic acid in the reaction sample can be cDNA, genomic DNA orviral DNA. However, the present invention can also be practiced withother nucleic acids, such as mRNA, ribosomal RNA, viral RNA. Thesenucleic acids may exist in a variety of topologies. For example, thenucleic acids may be single stranded, double-stranded, circular, linearor in form of concatamers. Those of skill in the art will recognize thatwhatever the nature of the nucleic acid, it can be amplified merely bymaking appropriate and well recognized modifications to the method beingused.

Detection of Amplified Target Nucleic Acid:

Amplified nucleic acids present in the subject chips may be detected bya range of methods including but not limited to (a) forming a detectablecomplex by, e.g., binding the amplified product with a detectable label;and (b) electrophoretically resolve the amplified product from reactantsand other components of the amplification reaction.

In certain embodiments, the amplified products are directly visualizedwith detectable label such as a fluorescent DNA-binding dye. Because theamount of the dye intercalated into the double-stranded DNA molecules istypically proportional to the amount of the amplified DNA products, onecan conveniently determine the amount of the amplified products byquantifying the fluorescence of the intercalated dye using the opticalsystems of the present invention or other suitable instrument in theart. DNA-binding dye suitable for this application include SYBR green,SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide,acridines, proflavine, acridine orange, acriflavine, fluorcoumanin,ellipticine, daunomycin, chloroquine, distamycin D, chromomycin,homidium, mithramycin, ruthenium polypyridyls, anthramycin, and thelike.

In addition to various kinds of fluorescent DNA-binding dye, otherluminescent labels such as sequence specific probes can be employed inthe amplification reaction to facilitate the detection andquantification of the amplified product. Probe based quantitativeamplification relies on the sequence-specific detection of a desiredamplified product. Unlike the dye-based quantitative methods, itutilizes a luminescent, target-specific probe (e.g., TaqMan® probes)resulting in increased specificity and sensitivity. Methods forperforming probe-based quantitative amplification are well establishedin the art and are taught in U.S. Pat. No. 5,210,015.

The subject chips and the associated optical systems are particularlysuited for conducting quantitative nucleic acid amplification.Accordingly, the present invention provides a method for monitoring theformation of a nucleic acid amplification reaction product, preferablyin real time. In certain preferred embodiments, the amplified nucleicacids contained are directly monitored by the photon-sensing elementsintegrated into the chips. The photon-sensing element registers theintensities of the optical signals that are reflective of the amount ofthe amplified nucleic acids at any time being examined during theamplification reaction. The optical signals may be any kind ofluminescent signals emitted upon exciting the labeled reactants withappropriate incident beams.

In another preferred embodiment, the amplified nucleic acids in thesubject chips are detected by the subject optical systems operativelycoupled to the chips. The optical systems are capable of transmittingappropriate excitation beams to the reactants in the amplificationreactions, collecting and analyzing the emitted optical signals from thereactants. Preferably, the optical signals detected are indicative ofthe amount of amplified nucleic acid in the amplification reaction overa multiple-cycle period. In certain aspects, the optical systemtransmits excitation beams into the wells containing the reactionsamples at a plurality of times during the amplification, and monitorsthe optical signals coming from the micro wells at each of the pluralityof times. By analyzing the relative intensities of the optical signals,preferably over a multiple-cycle period, one can monitor quantitativelythe progression of the amplification reaction. Typically, the opticalsignals being monitored are luminescent signals. In certain preferredaspects, detecting and/or monitoring the amplification products areperformed without opening the micro well once the amplification isinitiated.

Uses of Nucleic Acid Amplification and Detection Techniques of thePresent Invention:

The subject methods of amplifying and detecting a target nucleic acidhave broad spectrum of utility in, e.g. drug screening, diseasediagnosis, phylogenetic classification, genotyping individuals, parentaland forensic identification.

At a more fundamental level, amplification and detection of the targetnucleic acids may be used in identification and quantification ofdifferential gene expression between diseased and normal tissues, amongdifferent types of tissues and cells, amongst cells at differentdevelopmental stages or at different cell-cycle points, and amongstcells that are subjected to various environmental stimuli or lead drugs.

Other Chemical and Biological Applications:

The subject chips and other devices find utility in many other chemicaland biological applications where controllable temperatures are desired.Such applications include a vast diversity of reactions such as redoxreactions, hydrolysis, phosphorylation, and polymerization. Additionalapplications are directed to discerning interactions involvingbiological molecules such as proteins, glycoproteins, nucleic acids, andlipids, as well as inorganic chemicals, or any combinations thereof. Thechemical reaction may also involve interactions between nucleic acidmolecules, between nucleic acid and protein, between protein and smallmolecules The chemical reaction may take place outside a cell or insidea cell that is introduced into a micro well of the subject chip.

Of particular significance is the application in detecting the presenceof a specific protein-protein interaction. Such application generallyemploys a proteinaceous probe and a target protein placed in a microwell in the subject chip.

In one aspect of this embodiment, the protein-protein interaction isbetween a target protein (i.e. an antigen) and an antibody specific forthat target. In another aspect, the protein-protein interaction isbetween a cell surface receptor and its corresponding ligand. In yetanother aspect, the protein-protein interaction involves a cell surfacereceptor and an immunoliposome or an immunotoxin; in other aspects, theprotein-protein interaction may involve a cytosolic protein, a nuclearprotein, a chaperon protein, or proteins anchored on other intracellularmembranous structures.

The terms “membrane”, “cytosolic”, “nuclear” and “secreted” as appliedto cellular proteins specify the extracellular and/or subcellularlocation in which the cellular protein is mostly, predominantly, orpreferentially localized.

“Cell surface receptors” represent a subset of membrane proteins,capable of binding to their respective ligands. Cell surface receptorsare molecules anchored on or inserted into the cell plasma membrane.They constitute a large family of proteins, glycoproteins,polysaccharides and lipids, which serve not only as structuralconstituents of the plasma membrane, but also as regulatory elementsgoverning a variety of biological functions.

The reaction is typically performed by contacting the proteinaceousprobe with a target protein under conditions that will allow a complexto form between the probe and the target. The conditions such as thereaction temperature, the duration of the reaction, the bufferconditions and etc., will depend on the particular interaction that isbeing investigated. In general, it is preferable to perform thereactions under physiologically relevant temperature and bufferconditions. Physiologically relevant temperatures range fromapproximately room temperature to approximately 37° C. This can beachieved by adjusting the heating element of the subject chips.Typically, a physiological buffer contains a physiological concentrationof salt and at adjusted to a neutral pH ranging from about 6.5 to about7.8, and preferably from about 7.0 to about 7.5. A variety ofphysiological buffers is listed in Sambrook et al. (1989) supra andhence is not detailed herein.

The formation of the complex can be detected directly or indirectlyaccording standard procedures in the art or by methods describe herein.In the direct detection method, the probes are supplied with adetectable label and when a complex is formed, the probes emitted anoptical signal distinct from that of the unreacted probes. A desirablelabel generally does not interfere with target binding or the stabilityof the resulting target-probe complex. As described above, a widevariety of labels suitable for such application are known in the art,most of which are luminescent probes. The amount of probe-targetcomplexes formed during the binding reaction can be quantified bystandard quantitative assays, or the quantitative methods using theoptical systems described above.

Further illustration of the design and use of the chips according tothis invention is provided in the Example section below. The example isprovided as a guide to a practitioner of ordinary skill in the art, andis not meant to be limiting in any way.

Example 1

Amplification of a Target Nucleic Acid, Namely a Fragment of the G6PDHGene, is performed using a chip of the present invention. The reactionmixture containes G6PDH template, a pair of upstream and downstreamprimers specific for the template, dNTPs, and DNA polymerase.

The amplified products are detected with SYBR Green I that bindspreferentially to double-stranded DNA molecules (see, FIG. 20). FIG. 21depicts the SYBR Green staining of DNA molecules appeared at the threedifferent thermal stages of one PCR cycle. As is shown in FIG. 21,staining of the DNA is most intense at the annealing step (e.g., at 45°C.) because most of the DNA molecules assume a double helical structure.By contrast, very little SYBR Green staining is detected at thedenaturing step where the temperature is raised to e.g., 95° C. At about72° C. where primer-directed extension is taking place, a moderateamount of staining is detected. The amount of SYBR Green stain detectedthroughout one complete thermal cycle is quantified as shown in FIG. 22.

1-20. (canceled)
 21. A composition comprising: a chip comprising atleast 1000 fluidically isolated microwells, wherein said fluidicallyisolated microwells are at a surface density of 10 or more microwellsper cm².
 22. The composition of claim 21, wherein said fluidicallyisolated microwells are at a surface density of 100 or more microwellsper cm².
 23. The composition of claim 21, wherein said fluidicallyisolated microwells are at a surface density of from 5-50 microwells permm².
 24. The composition of claim 21, wherein said fluidically isolatedmicrowells are at a surface density of at 1 or more microwell per mm².25. The composition of claim 21, wherein said chip has a length from afew millimeters to 50 centimeters.
 26. The composition of claim 21,wherein said chip has a width from a few millimeters to 50 centimeters.27. The composition of claim 21, wherein said fluidically isolatedmicrowells have a volume of 1 μL or less.
 28. The composition of claim21, wherein said fluidically isolated microwells have a volume rangingfrom 0.001 μL to 100 μL.
 29. The composition of claim 21, wherein saidchip comprises a material selected from the group consisting of:silicon, silicates, gallium phosphide, glass, ceramic, metals, andalloys, or any combination thereof.
 30. The composition of claim 21,wherein said chip comprises aluminum alloy.
 31. The composition of claim21, wherein said chip has a thermal conductivity value of 1 W/mK orgreater.
 32. The composition of claim 21, wherein said fluidicallyisolated microwells are coated with a hydrophobic polymer.
 33. Thecomposition of claim 21, wherein said fluidically isolated microwellsare arrayed in a grid.
 34. The composition of claim 21, wherein saidfluidically isolated microwells comprise a length, width, or depthranging from 10 millimeter to 100 micrometer in length.
 35. Thecomposition of claim 21, wherein the transverse sectional area of saidfluidically isolated microwells is selected from the group consistingof: circular, elliptical, oval, conical, rectangular, triangular, andpolyhedral, or any combination thereof.
 36. The composition of claim 21,further comprising a heating element in thermal contact with a base ofsaid fluidically isolated microwells.
 37. The composition of claim 21,further comprising a heating element in thermal contact with a top ofsaid fluidically isolated microwells.
 38. The composition of claim 21,wherein said fluidically isolated microwells comprise reagents fornucleic acid amplification.
 39. The composition of claim 21, furthercomprising a dispensing system operatively linked to said chip.
 40. Thecomposition of claim 21, wherein said chip is sealed.